Foamy Viruses (FVs; Spumavirinae) are classified as retroviruses and demonstrate classic retroviral genomic organization, including the three hallmark genes gag, pol, and env (FIG. 1; Rethwiln, Curr. Top. Microbiol. Immunol. 193:1-24 (1995)). Despite this classification and organization, several aspects of the FV life cycle differ significantly from retroviruses. Three of these differences highlight unique properties of the polymerase (Pol) protein and the reverse transcriptase (RT) enzyme encoded within it. First, FV has an unusual mechanism for the expression of Pol. Typical retroviruses express Pol as part of a Gag-Pol fusion protein, which mediates Pol incorporation into the virion through Gag-Gag interactions. In contrast, FV Pol is expressed from its own spliced message, and consequently FV must employ a unique strategy for incorporation of Pol into the viral particle (Lochelt and Flugel, J. Virol. 70:1033-1040 (1996), Yu et al. Science 271:1579-1582 (1996)). Second, reverse transcription occurs at a different stage in the FV life cycle. Unlike conventional retroviruses, FV particles contain DNA that appears to be used as the functional genome when infecting a new cell (Moebes et al., J. Virol. 71:7305-7311 (1997), Yu et al., J. Virol. 73:1565-1572 (1999)). This requires that FV RT be active during, or shortly after particles bud from an infected cell. Third, the FV Pol polyprotein undergoes limited processing. A single cleavage event between RT and Integrase (IN) takes place resulting in two mature enzymatic proteins, IN and a PR-RT fusion protein (Pfrepper et al., Virus Genes 22:61-72 (2001)).
Because of its central role in the retroviral life cycle, RT has been a target of drugs designed to inhibit HIV-1 replication and control infections. One major class of these RT inhibitors is the nucleoside analog inhibitors, or chain terminators, which include (AZT) and (3TC). In vitro, these drugs have proven to be potent inhibitors of RT activity and viral replication for many retroviruses, including HIV-1. However, it has been demonstrated previously that of 3TC, AZT, and ddI, only AZT specifically inhibits SFVcpz (hu), (a human foamy virus newly designated Prototype Foamy Virus (PFV)), replication (Yu et al., J. Virol. 73:1565-1572 (1999)).
In HIV-1 patients, these drugs have been shown to inhibit viral replication, but HIV-1 is able to rapidly evolve mutations that allow it to overcome this inhibition. In the case of 3TC, resistance rapidly and consistently develops through mutation of the second residue in the HIV-1 catalytic motif Tyr Met Asp Asp (YMDD) to valine (Tisdale et al. Proc. Natl. Acad. Sci. USA 90:5653-5656 (1996)). The YXDD (SEQ ID NO: 17) catalytic motif, wherein X defines any amino acid residue, is highly conserved among all reverse transcriptases, and all retroviral RTs contain a methionine in the second residue with the exception of Murine Leukemia Viruses (MLVs) and FVs, which contain a valine in this position. When the amino acid sequence of SFVcpz(hu) RT (PFV RT) is compared to other retroviral RTs, the degrees of homology range from about 27% (HIV-2) to about 34% (Mo-MLV) (Maurer et al. J. Virol. 62:1590-1597 (1988)). Despite this low level of overall homology, PFV RT contains residues shown to be functionally essential in all other retroviral RTs, including the YXDD motif (SEQ ID NO: 17). Interestingly, both MLV and FV contain a valine in their catalytic YXDD motifs (SEQ ID NO: 17), and both are naturally resistant to 3TC.
In the context of the virus, RT is expressed as part of a larger Pol polyprotein, which also contains Integrase (INT) and Protease (PR). Unlike other retroviruses, the FV Pol undergoes a single cleavage event to release INT, leaving a mature PR-RT fusion protein. However, in bacterial over-expression systems a second cleavage event between PR and RT has been previously observed. To avoid this artificial cleavage, as provided herein the entire PR-RT fusion protein was bacterially expressed with a point mutation in the PR active site termed D/A, wherein an alanine replaces an aspartic acid residue. Both wild type and a mutated (V313M) RTs were successfully expressed and purified in the D/A context.
Further, it was determined from mutations in the RT which reduce the activity of the RT by about 45 to 60% that foamy virus (FV) production was inhibited or stopped when RT activity was reduced. Therefore, unlike other retroviruses FV requires a highly active reverse transcriptase. It has also been determined by the present invention that FV requires a reverse transcriptase that is highly processive, i.e., capable of producing long nucleotide transcription products. In one embodiment of the present invention an isolated FV RT has been found to be capable of generating products that were well above 600 base pairs(bp) in 10 minutes. In similar studies the processivity of HIV-1 reverse transcriptase has been measured to generate transcript products of 150 bp or smaller (Boyer and Hughes, Microbiol. Agents Chemother. 39:1624-8 (1995)).